Separation of rat plasma HDL subfractions by density gradient centrifugation and the effect of incubation on these fractions

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11-19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.     

Intracellular distribution of fumarase in various animals

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.     

Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat

Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 +/- 2.8, 62.4 +/- 4.5, and 224 +/- 16 mM/s) and exceeded the NMR-determined in vivo reaction velocities by an order of magnitude (4.1 +/- 1.2, 5.1 +/- 1.6, and 18.4 +/- 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues: greater than 99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98% MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p less than 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.     

Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis

Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, functional, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and α-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles.     

Differential Effect of Denervation on Free-Radical Scavenging Enzymes in Slow and Fast Muscle of Rat

To determine the effect of denervation on the free-radical scavenging systems in relation to the mitochondrial oxidative metabolism in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, the sciatic nerve of the rat was crushed in the midthigh region and the muscle tissue levels of five enzymes were studied 2 and 5 weeks following crush. Recently developed radioimmunoassays were utilized for the selective measurement of cuprozinc (cytosolic) and mangano (mitochondrial) superoxide dismutases. Total tissue content of cuprozinc superoxide dismutase showed a mild decrease after denervation in slow but not in fast muscle. Manganosuperoxide dismutase and fumarase decreased markedly at 2 weeks and returned toward control levels by 5 weeks, the changes appearing to be greater in slow than in fast muscle. At 2 weeks, cytochrome c oxidase decreased significantly in slow, but not in fast muscle. GSH-peroxidase at baseline was 10-fold higher in slow than in fast muscle, markedly decreased at 2 weeks in slow muscle, and returned toward control levels at 5 weeks, whereas the total enzyme activity in fast muscle did not change through 5 weeks. These data represent the first systematic report of free radical scavenging systems in slow and fast muscles in response to denervation. Selective modification of cuprozinc and manganosuperoxide dismutases and differential regulation of GSH-peroxidase was demonstrated in slow and fast muscle.     

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.     

Modulation of mitochondrial nitric oxide synthase and energy expenditure in rats during cold acclimation

To preserve thermoneutrality, cold exposure is followed by changes in energy expenditure and basal metabolic rate (BMR). Because nitric oxide (NO) modulates mitochondrial O(2) uptake and energy levels, we analyzed cold effects (30 days at 4 degrees C) on rat liver and skeletal muscle mitochondrial NO synthases (mtNOS) and their putative impact on BMR. Cold exposure delimited two periods: A (days 1-10), with high systemic O(2) uptake and weight loss, and B (days 10-30), with lower O(2) uptake and fat deposition. mtNOS activity and expression decreased in period A and then increased in period B by 60-100% in liver and skeletal muscle (P < 0.05). Conversely, mitochondrial O(2) uptake remained initially high in the presence of l-arginine and later fell by 30-50% (P < 0.05). On this basis, the estimated fractional contribution of liver plus muscle to total BMR varied from 40% in period A to 25% in period B. The transitional modulation of mtNOS in rat cold acclimation could participate in adaptive responses that favor calorigenesis or conservative energy-saving mechanisms.     

Fumarase activity in skeletal muscle of man

Soluble extracts of human skeletal muscle have a fumarase activity of 31.2 +/- 7.27 (M. vastus lateralis quadricipitis) or 30.9 +/- 8.0 U/g wet weight at 37 degrees C (M. deltoideus). The distribution of activities in the 36 muscle samples studies is not gaussian. There is a significant correlation between the activities of citrate synthase and fumarase (r = + 0.881; p less than 0.001) in all investigated muscles, excepting M. vastus lateralis quadricipitis.     

Isolation of the cDNA encoding rat skeletal muscle myosin light chain kinase. Sequence and tissue distribution

A cDNA clone encoding skeletal muscle myosin light chain kinase (MLCK) was isolated from a rat skeletal muscle library using oligonucleotide probes. The total length of the rat skeletal muscle MLCK cDNA was 2823 base pairs with an open reading frame of 1830 base pairs. The deduced sequence of the 610-amino acid protein exhibited 96% amino acid identity to rabbit skeletal muscle MLCK in the carboxyl-terminal portion of the molecule, which contains the catalytic and the calmodulin-binding domains, and 58% identity in the amino-terminal region. Analysis of total rat mRNA revealed a single mRNA species of 3.4 kilobases that was unique to skeletal muscle. Further analysis of skeletal muscle tissue using fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-twitch oxidative fibers isolated from rat leg revealed that the mRNA level for MLCK varied among the three fiber types. The results of kinase assays performed on the fibers showed that MLCK activity levels paralleled the MLCK mRNA levels found in each of the three types of skeletal muscle fibers studied. Fast-twitch oxidative glycolytic (gastrocnemius red) and slow-twitch oxidative (soleus) exhibited 60 and 13%, respectively, of the enzymatic activity present in fast-twitch glycolytic (gastrocnemius white) fibers.     

Intracellular distribution of NADP-linked isocitrate dehydrogenase,fumarase and citrate synthase in bovine heart muscle

By fractional extraction of minced bovine heart muscle with iso-osmotic sucrose and phosphate buffer solutions, it is shown that less than 4% of the total citrate synthase in the tissue is in the cytosol. Using citrate synthase as a marker for broken mitochondria, two methods of fractionation of 750 × g supernatants from homogenates of bovine heart muscle show that 10% of the total fumarase and NADP-linked isocitrate dehydrogenase activities are present in the cytoplasm. Homogenates prepared by sonication and osmotic shock and by sand-grinding gave closely similar results as regards enzyme distributions and extent of mitochondrial breakage. The results are compared with those reported for other tissues.     

Identification and characterization of a tissue kallikrein in rat skeletal muscles.

A tissue kallikrein was purified from rat skeletal muscle. Characterization of the enzyme showed that it has alpha-N-tosyl-L-arginine methylesterase activity and releases kinin from purified bovine low-Mr kininogen substrate. The pH optimum (9.0) of its esterase activity and the profile of inhibition by serine-proteinase inhibitors are identical with those of purified RUK (rat urinary kallikrein). Skeletal-muscle kallikrein also behaved identically with urinary kallikrein in a radioimmunoassay using a polyclonal anti-RUK antiserum. On Western-blot analysis, rat muscle kallikrein was recognized by affinity-purified monoclonal anti-kallikrein antibody at a position similar to that of RUK (Mr 38,000). Immunoreactive-kallikrein levels were measured in skeletal muscles which have different fibre types. The soleus, a slow-contracting muscle with high mitochondrial oxidative-enzyme activity, had higher kallikrein content than did the extensor digitorum longus or gastrocnemius, both fast-contracting muscles with low oxidative-enzyme activity. Streptozotocin-induced diabetes reduced muscle weights, but did not alter the level of kallikrein (pg/mg of protein) in skeletal muscle, suggesting that insulin is not a regulator of kallikrein in this tissue. Although the role of kallikrein in skeletal muscle is unknown, its localization and activity in relation to muscle functions and disease can now be studied.     

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.     

Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.     

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in heart and muscle phosphofructokinase isozymes

Phosphofructokinase isozymes of fetal, neonatal, and adult rat heart and skeletal muscle were characterized by DEAE-cellulose chromatography, agarose gel electrophoresis, and immunodiffusion with specific antisera. The results of these studies indicate that in skeletal muscle and heart the levels of the major liver phosphofructokinase isozyme (PFK-L2) and the muscle phosphofructokinase isozyme (PFK-M) are dependent on the developmental status of the rat. For example, PFK-L2 and PFK-M are present in fetal and early neonatal skeletal muscle; whereas in adult skeletal muscle, only PFK-M is detectable. By DEAE- cellulose chromatography, PFK-L2 activity was estimated to be 2.4 units/g (41% of total phosphofructokinase activity) in fetal muscle, very low and not resolved from PFK-M in 7-day neonatal muscle, and not detectable in adult muscle. Further, PFK-M activity was found to be 3.4 units/g (59% of total phosphofructokinase activity), 10 units/g, and 31.6 units/g in fetal, 7-day neonatal, and adult skeletal muscle, respectively. The developmental changes of heart phosphofructokinase isozymes differ considerably from that of the skeletal muscle phosphofructokinase isozymes. In fetal heart, PFK-L2 is the major phosphofructokinase isozyme (5.6 units/g), constituting 67% of total phosphofructokinase activity. Further, in fetal heart another phosphofructokinase isozyme (33% of total phosphofructokinase activity) was found by DEAE-cellulose chromatography which is different from PFK-M and PFK-L2. In 7-day neonatal and adult heart, PFK-M and PFK-L2 are the only detectable phosphofructokinase isozymes. Varying from 5.6 units/g (44% of total) in 7-day neonatal to 5.9 units/g (40% of total) in adult heart, PFK-L2 activity remains fairly constant. Also, PFK-M is very low in fetal heart but increases within 1 week postpartum to 5.5 units/g (50% of total activity) and to 8.9 units/g (60% of total activity) in adult heart.     

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization

The distribution of glycogen synthase (casein) kinase-1 (CK-1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK-1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK-1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK-1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK-1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK-1 may be involved in the regulation of diverse cellular functions.     

Comparison of populations of mRNA coding fumarase in rat brain and liver

The populations of mRNA encoding mitochondrial and cytosolic fumarases in the poly A+ RNA fractions purified from polysomes of rat brain and liver were examined. When the in vitro translation products programmed by the poly A+ RNA fraction obtained from rat brain were purified by immunoprecipitation with anti-fumarase antibody and analyzed by SDS polyacrylamide gel electrophoresis and fluorography, only one polypeptide of 50 KD was detected as a precursor of fumarase. In contrast, by the same method, two polypeptides of 50 KD and 45 KD, which is the same size as mature fumarase, were detected as precursors of rat liver fumarase. These results suggest that rat brain polysomes contain only one population of mRNA coding a 50 KD precursor of mitochondrial fumarase with little or no mRNA of the cytosolic fumarase, whereas rat liver polysomes contain two types of mRNA for mitochondrial and cytosolic fumarases, respectively. These findings are consistent with the fact that the brain is the only organ in rats known not to contain cytosolic fumarase.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O₂ consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.